Messenger RNA levels in eukaryotic cells are regulated by proteins that determine which genes are transcribed and precisely govern RNA polymerase II activity. These transcription factors usually contain both DNA-binding and activation domains. They function by recognizing a specific DNA sequence in a particular gene's promoter, or control region, and then present their activation domains to the polymerase and its accessory proteins, known as general transcription initiation factors. This second molecular recognition event is thought to stabilize the transcription machinery on a given promoter, increasing the efficiency of RNA production from that gene. Among the best characterized of these transcriptional activators are members of the family of helix-loop-helix proteins, which bind their DNA targets as homo- and!or heterodimers and regulate mRNA production from a host of critical cellular genes, sometimes acting as oncogenes causing malignancy. These proteins also form oligomers (tetramers) that are bivalent, possibly mediating DNA looping and affecting mRNA synthesis. DNA binding, homo- and heterodimerization and tetramerization are supported entirely by the so-called helix-loop-helix domains of these proteins, which are similar in amino acid sequence and typically consist of a basic region, a helix-loop-helix region and sometimes a leucine heptad repeat or zipper region. We have previously determined the structures of the Max and upstream stimulatory factor homodimers bound to their target DNA at modest resolution, characterized the specific random coil to alpha-helix folding transition that occurs on DNA binding, and demonstrated that USF can function as a bivalent homotetramer binding two recognition sites simultaneously. The goal of the proposed research is to study the DNA-binding domains of selected helix-loop-helix transcription factors (Max, Myc, Mad, upstream stimulatory factor, hairy, CBF1 achaete scute, AP-2) with synchrotron radiation/cryocrystallography and analytical ultracentrifugation, and derive a comprehensive structural and functional understanding of DNA recognition, homo- and heterodimerization specificity, tetramerization, the role of the loop region, and their respective effects on mRNA production. High-resolution cocrystal forms of Max and upstream stimulatory factor homodimers are already in hand, and small cocrystals have been obtained with the hairy homodimer. Covalent heterodimers of Myc-Max and Mad-Max have been prepared by total chemical synthesis and full native DNA-binding activity has been demonstrated for the synthetic Myc-Max heterodimer, which is analogous to the active form o Myc that causes malignancy. Ultracentrifugation studies have been performed with the homomeric forms of both upstream stimulatory factor and Max.